Large-conductance, voltage- and Ca2+-activated BK potassium channels are regulators of membrane excitability and of cytoplasmic Ca2+. These channels are opened by two additive inputs, a depolarizing change in membrane potential, which activates the voltage-sensors, and an increase in [Ca2+]IN, which increases occupation of the Ca2+ binding sites. These inputs are linked through propagated changes in the structure of the channel complex, a mechanism well-described by an allosteric kinetic model. The different structures in the activated and deactivated states of the voltage-sensor domains, in the occupied and unoccupied states of the Ca2+-binding domains, and more broadly in the open and closed states are stabilized by interactions that must differ to some extent in the amino acid residues involved. These interactions that differ in the different states are the pistons and gears of the machine. This is true of the tetrameric complex of BK a which alone forms a voltage- and Ca2+-gated channel. This is also true of the complex of four a and four 1, in which the 1 subunits modulate the function of the a subunits. 1 acts as a ligand of a, and its binding site changes in the different functional states of a. The binding interface is likely to be extensive and discontinuous. The long-term goals of this project are to characterize at the residue level the interface between a and 1 and to identify those interactions that change with the change in functional state. We propose to accomplish these goals by determining the pairwise proximities between a large number of substituted cysteines (Cys), based on their extents and rates of disulfide bond formation in the closed and open states of the BK channel, and by using these proximities as constraints in modeling the BK channel a and 1 complex in its different functional states. This would be impractical except that the channel-forming and voltage-sensing part of the BK channel a subunit, formed by its transmembrane (TM) helices S1-S6, is homologous to the chimeric Kv1.2/Kv2.1 channel, for which there is a high-resolution structure. In addition, BK a contains a seventh TM helix, S0, for which there is no precedent. Moreover, there are no 3D structures of any of the four types of subunits. S0 and 1, however, are small enough that their interactions with the conserved S1-S6 domain can be usefully characterized by medium-resolution methods and by modeling. We propose to substitute, systematically and extensively, pairs of cysteines and 1) to rank the proximities of the Cys based on their extents and rate constants of disulfide bond formation, 2) to determine the functional consequences of crosslinking, and conversely 3) to determine whether crosslinking is dependent on functional state. We have used this approach successfully to locate the extracellular ends of S0, and of the two TM helices of 1, TM1 and TM2, relative to each other and to S1-S6, in the tetrameric channel structure. We now propose to do the same with the intracellular ends of a S0 and of 1 TM1 and TM2 and their intracellular N- and C-terminal tails. PUBLIC HEALTH RELEVANCE: The large-conductance, voltage- and Ca2+-activated BK potassium channels play key roles in many different cell types in the control of excitability and cytoplasmic calcium concentration. Gain-in-function mutations in BK channels are associated with protection from, and loss-of-function mutations are associated with increased incidence of, hypertension, asthma, and epilepsy. Thus, the proposed research is relevant to public health and to the NIH mission to discover basic mechanisms underlying human disease.